Arteriosclerosis, which literally means "hardening of the arteries," actually refers to a group of disorders which involves a thickening and loss of arterial elasticity. Although they frequently occur together, each of the principal disorders (Monckeberg's medial calcific sclerosis, arteriolosclerosis, and atherosclerosis) are distinguishable by the afflicted artery's morphological appearance. Monckeberg's medial calcific sclerosis is characterized by ringlike calcifications in small to medium sized arteries. Arteriolosclerosis is characterized by a thickening of artery and arteriole walls, resulting in lumen narrowing.
The predominant and most serious form of arteriosclerosis is atherosclerosis. In Western countries, atherosclerosis is responsible for 20% to 25% of myocardial infarction deaths yearly, and is a contributing factor in about 50% of deaths from all other causes. Atherosclerosis is also the major cause of a large number of morbidities, including chronic ischemic heart disease, gangrene, mesenteric occlusion, and ischemic encephalopathy.
Atherosclerosis is believed to begin in childhood as a progressive disease that first strikes the large- and medium-sized arteries. These include the coronary, the carotid, the aorta, and the larger arteries of the lower extremities. Later in life, lesions called "atheromas" or "fibrofatty plaques" form on the arterial inner walls. These plaques have a central necrotic core of lipid deposits composed primarily of cholesterol, calcium, cellular debris, and other materials. As the disease progresses, plaques coalesce, forming large masses, or what have been called "complicated plaques," in which there may be associated arterial calcification. Fatty streaks appear on the vessel walls, causing them to ulcerate and rupture. Debris (such as cholesterol emboli) are then released into the bloodstream. Hemorrhaging may occur from rupture of the overlying capillary endothelium. Eventually, clotting may occur within the vessel, causing tissue infarction.
The major risk factors associated with heart disease include hypercholesterolemia, hypertension, hyperglycemia and smoking. Hypercholesterolemia (high serum cholesterol) is present most of the time, and serum cholesterol concentrations correlate with mortality. The death rate from cardiac disease is three times greater in men with serum cholesterol concentrations greater than about 200 milligrams per deciliter (mg/dl) as compared to individuals having lower cholesterol concentrations in the same age groups.
There is a direct link between hypercholesterolemia and the incidence of complicated plaque formation and atherosclerosis. Atherosclerotic plaques are rich in cholesterol, particularly cholesterol esters. Experimental animals fed diets high in cholesterol or that have genetic disorders which produce high plasma cholesterol levels develop full-blown atherosclerosis. In humans, it is clear that a direct relationship exists between plasma cholesterol concentrations and mortality. Individuals who consume a diet rich in cholesterol and saturated fat have elevated plasma cholesterol concentrations and a higher incidence of atherosclerosis. Conversely, high risk individuals who take cholesterol-lowering drugs and restrict cholesterol intake have a lower incidence of cardiovascular mortality.
All plasma lipids, including cholesterol and triglyceride, circulate through the bloodstream in association with proteins. The major cholesterol- and triglyceride-carrying proteins are a group of lipoproteins collectively referred to as apoproteins. Coupled with lipid cargo, these lipoprotein complexes are classified according to their flotation constants or densities. Some of the major lipoprotein complexes include high density lipoprotein (HDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), and chylomicron. LDLs carry the primary plasma cholesterol load, and high amounts of the LDLs and plasma triglycerides are associated with an increased risk of atherosclerosis and related cardiovascular diseases.
Humans need only small amounts of cholesterol. Most cholesterol is produced in the liver, the only organ capable of breaking down and excreting excess cholesterol. The body's capacity to break down and rid itself of excess cholesterol is limited.
Excess cholesterol is carried by lipoprotein particles. These cholesterol-laden particles find small cracks in arterial walls, and initiate a process leading to fatty fibrous plaque buildup and arterial lumen obstruction. In coronary artery disease, lumen obstruction restricts blood flow. Lack of oxygen and other nutrients leads to cardiac muscle necrosis.
Coronary artery disease is the most common cause of death in the United States. Approximately thirty million people suffer from coronary artery disease, and nearly one million die from it each year.
Recent studies suggest a link between serum cholesterol reduction and arterial plaque reversal. The 1987 Cholesterol Lowering Atherosclerosis Study (CLAS) showed arterial plaque reversal in 16% of treated subjects as compared to 2% of placebo subjects. The treated subjects were given a combination of first-generation cholesterol control agents. Disease progression continued in 39% of the treated group and in 60% of the placebo group.
Presently, 85% of the cholesterol control agent market is covered by a class of drugs known as HMG Co-A reductase inhibitors. These drugs inhibit hepatic cholesterol synthesis. However, they have no effect on dietary cholesterol. All classes of cholesterol-lowering drugs are indicated for therapeutic use in familial hypercholesterolemia, a disorder that affects less than 1.5% of the population. All require a strict dietary regimen to achieve a significant lipid-lowering effect. These drugs may cause unwarranted side effects and have significant contra-indications. Side effects include rhabdomyolysis, myopathy, cataracts, and lupus erythematosus. Drug use is contraindicated in pregnancy and in liver disease.
For these reasons, the FDA and many clinicians have been reluctant to support the use of these agents for atherosclerosis prophylaxis (prevention), even in "high-risk" (Group I) patients, such as post-coronary bypass subjects. This attitude is changing however, and more physicians are treating "potentially high risk" (Group II) patients with cholesterol control agents. Therapeutic intervention could also be helpful as a prophylactic in "low risk" (Group III) individuals.
Several large population studies are being conducted with cholesterol synthesis control agents. The Healthy Heart Study in the United Kingdom will recruit eighteen thousand patients from general practitioners across the country. Over the next five years, some individuals will be given cholesterol synthesis lowering agents, and some individuals will be given placebos. The chief aim of this study is to determine the effects of cholesterol on mortality rates in a large population. The West of Scotland Coronary Prevention Study (WOSCOPS) will evaluate the effects of PRAVASTATIN on coronary artery disease in six thousand men.
These studies will establish the precedent for using cholesterol-lowering agents prophylactically. By treating Group III "low risk" individuals, the number of potential users in the U.S. could reach seventy to eighty million people by the end of the decade. Treatment of low risk populations in Europe, Latin America and the Orient could increase use to two hundred million people worldwide.
The cholesterol pool in an organism is primarily derived from two sources: 1) absorption of dietary cholesterol and 2) de novo synthesis. The size of the cholesterol pool can be decreased by restricting synthesis, restricting dietary intake, or excreting cholesterol through its conversion to bile acids. As discussed above, the prior art describes several agents for decreasing de novo cholesterol synthesis. Usually, reduction of dietary cholesterol is also required to achieve the desired plasma cholesterol levels.
An alternative strategy is to increase the body's ability to excrete cholesterol by converting cholesterol to bile acids. This method has the advantage of lowering serum cholesterol concentrations, irrespective of cholesterol origin (diet or synthesis). Thus, strict low-cholesterol diets may not be required if cholesterol can be effectively removed. Elimination of cholesterol by its conversion to bile acids also avoids any undesired effects associated with inhibiting steroid synthesis. However, in humans, rabbits, and other animals that develop hypercholesterolemia, cholesterol and oxysteroids inhibit cholesterol conversion to bile acids. Thus, serum cholesterol concentrations increase.
Molowa, D. T., et al., "Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene," Biochemistry 31:2539-2544 (1992) and Takiguchi, Japanese Patent Application No. W092/00088 report non-specific proteins that stimulate bile acid production. However, the known methods for increasing bile acid production lack specificity and are known to affect other enzyme systems. The prior art does not disclose a specific agent which increases conversion of cholesterol to bile acids without affecting other enzyme systems.
Primary bile acids are synthesized in hepatocytes directly from cholesterol. The major bile acids in humans and some other mammals are cholic acid and chenodeoxycholic acid. Primary bile acids are often conjugated with glycine or taurine to form glycocholic or taurocholic acids, respectively. Primary bile acids undergo metabolism by intestinal flora to secondary bile acids such as deoxycholic and lithocholic acid.
Bile acids play a significant role in cholesterol metabolism. They represent the most significant pathway by which cholesterol and similar compounds bearing a steroid ring structure are excreted. In bile, bile acids prevent the precipitation of cholesterol and similar compounds in the gallbladder. Bile acids act as emulsifying agents and have a role in preparing triglycerides for lipase cleavage. Bile acids also facilitate absorption of fat-soluble vitamins across the digestive tract.
The rate limiting step in conversion of cholesterol to bile acids involves hydroxylation of carbon number 7 by cholesterol 7-alpha-hydroxylase (EC 1.14.13.17) ("C7.alpha.H"), which comprises a microsomal monooxygenase cytochrome P-450.sub.C7.alpha.H and a NADPH-cytochrome P-450 reductase. Cholesterol elimination through this pathway is important in controlling disorders such as, but not limited to, arteriosclerosis, hyperlipidemia, hypercholesterolemia, gallstone disease, and lipid storage diseases.
The cloning of a cDNA encoding human cytochrome P-450.sub.C7.alpha.H was disclosed in Noshiro, M. and Okuda, K., "Molecular cloning and sequence analysis of cDNA encoding human cholestersol 7 alpha-hydroxylase," FEBS 268:137-140 (1990), and the gene for rat C7.alpha.H in Nishimoto, M., et al., "Structural analysis of the gene encoding rat cholesterrol alpha-hydroxylase, the key enzyme for bile acid synthesis," J. Biol. Chem. 266:6467-6469 (1991). Jelinek, D. F., et al., J. Biol. Chem. 265:8190-8197 (1990), and Li, Y. C., et al., "Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing and regulation of cholesterol 7 alpha-hydroxylase mRNA," J. Biol. Chem. 265:12012-12019 (1990) also disclose C7.alpha.H cDNA sequences. However, none of these references disclose the particular nucleotide sequence taught in the present invention, nor do they disclose specific regulatory factors which control expression of C7.alpha.H (C7.alpha.H regulatory factor) or related factors for controlling cholesterol or other steroids. Molowa, D. T., et al., "Transcriptional regulation of the human cholesterol 7 alpha-hydroxylase gene," Biochemistry 31:2539-2544 (1992), suggest that hepatocyte nuclear factor-3 ("HNF-3") may be involved in the control of C7.alpha.H expression. However, the C7.alpha.H regulatory factors of the present invention are not the HNF-3 factors of Molowa, D. T., et al., as shown in Example 7, below.
The prior art provides no mechanism for avoiding the inhibition of C7.alpha.H expression by increased levels of cholesterol which occurs in species susceptible to hyperlipidemia and atherosclerosis. The art does not teach a C7.alpha.H promoter sequence which interacts with a regulatory factor specifically regulating C7.alpha.H expression in a manner that reduces the inhibition of cholesterol conversion to bile acids by increased cholesterol concentrations. These and other shortcomings of the prior art are overcome by the present invention as described below.